Antibiotic A-32887

ABSTRACT

Antibiotic A-32887 is produced by submerged aerobic fermentation of Streptomyces albus NRRL 11109. A-32887 is an antibacterial, antiprotozoal, anticoccidial, and insecticidal agent. A-32887 also increases feed-utilization efficiency in ruminants.

This is a division of application Ser. No. 801,876, filed May 31, 1977.

BACKGROUND OF THE INVENTION

1. Field of the Invention

New, improved antibiotics are continually in demand. In addition to antibiotics which are useful for human diseases, improved antibiotics are also needed in the veterinary field. Improved growth promotion in animals is one important goal for these antibiotics. Growth promotion is achieved, for example, by reducing disease and by increasing feed-utilization efficiency.

Coccidiosis is one disease important to veterinary science, especially to the poultry industry. Coccidiosis results from infection by one or more species of Eimeria or Isopora (for a summary see Lund and Farr in "Diseases of Poultry," 5th ed., Biester and Schwarte, Eds., Iowa State University Press, Ames, Iowa, 1965, pp 1056-1096). Economic losses due to coccidiosis are great, and known anticoccidial agents have many disadvantages. Improved anticoccidial agents continue to be needed.

Promotion of growth in ruminants, such as cattle, is another economically-desirable objective of veterinary science. Of particular interest is growth promotion which is achieved by increasing feed-utilization efficiency. The mechanism for utilization of the major nutritive portion (carbohydrates) of ruminant feeds is well known. Microorganisms in the rumen of the animal degrade carbohydrates to produce monosaccharides and then convert these monosaccharides to pyruvate compounds. Pyruvates are metabolized by microbiological processes to form acetates, butyrates or propionates, collectively known as volatile fatty acids (VFA). For a more detailed discussion, see Leng in "Physiology of Digestion and Metabolism in the Ruminant," Phillipson et al., Eds., Oriel Press, Newcastle-upon-Tyne, England, 1970, pp 408-410.

The relative efficiency of VFA utilization is discussed by McCullough in Feedstuffs, June 19, 1971, page 19; Eskeland et al. in J. An. Sci. 33, 282 (1971); and Church et al. in "Digestive Physiology and Nutrition of Ruminants," Vol. 2, 1971, pp 622 and 625. Although acetates and butyrates are utilized, propionates are utilized with greater efficiency. A beneficial compound, therefore, stimulates animals to produce a higher proportion of propionates from carbohydrates, thereby increasing carbohydrate-utilization efficiency.

2. The Prior Art

A-32887 is a new member of the group of polyether antibiotics. Examples of members of this group include monensin (U.S. Pat. No. 3,501,568); dianemycin [R. L. Hamill, M. M. Hoehn, G. E. Pittenger, J. Chamberlin, and M. Gorman, J. Antibiotics 22, 161 (1969)]; nigericin [L. K. Steinrauf, Mary Pinkerton, and J. W. Chamberlin, Biochem. Biophys. Res. Comm. 33, 29 (1968)]; salinomycin (U.S. Pat. No. 3,857,948); A-130-A (U.S. Pat. No. 3,903,264); A-28695 A and B (U.S. Pat. No. 3,839,558); grisorixin [Chem. Commun., p. 1421 (1970)]; A-218 and K-41 [J. Antibiotics 29 (1), 10-14 (1976)].

Of this group of antibiotics, A-32887 is most closely related to K-41. A convenient method for distinguishing A-32887 from K-41 is by chromatography. A-32887 can be separated from K-41, for example, by silica-gel thin-layer chromatography in the following two systems (R_(f) values are approximate):

    ______________________________________                                         Antibiotic  Solvent System   R.sub.f Value                                     ______________________________________                                         A-32887     chloroform:methanol                                                                             0.78                                                          (92:8)                                                             K-41        chloroform:methanol                                                                             0.84                                                          (92:8)                                                             A-32887     ethyl acetate:ethanol                                                                           0.76                                                          (1:4)                                                              K-41        ethyl acetate:ethanol                                                                           0.70                                                          (1:4)                                                              ______________________________________                                    

SUMMARY OF THE INVENTION

This invention relates to an antibiotic substance which is produced by culturing a hitherto undescribed strain of the organism Streptomyces albus NRRL 11109.

The antibiotic substance of this invention is arbitrarily designated herein as A-32887. The C₂ -C₆ -acyl ester derivatives of antibiotic A-32887, the methyl ether derivative of antibiotic A-32887, and the pharmaceutically-acceptable salts of antibiotic A-32887 and of said ester and ether derivatives are also part of this invention. To simplify discussions of utility, the term "A-32887 compound" is used and refers to antibiotic A-32887, a specified acyl ester derivative of A-32887, the methyl ether derivative of A-32887 or a pharmaceutically-acceptable salt of A-32887 or of said ester or ether derivatives.

A-32887 is produced by culturing a novel strain of Streptomyces albus, NRRL 11109, under submerged aerobic fermentation conditions until a substantial level of antibiotic activity is produced. A-32887 is extracted from basified broth filtrate with polar organic solvents. The extracted A-32887 is purified by absorption chromatography.

A-32887 inhibits the growth of organisms which are pathogenic to animal and plant life. More specifically, A-32887 is an antibacterial, antifungal, antiprotozoal, anticoccidial, antiviral and insecticidal agent. In addition, A-32887 increases feed-utilization efficiency in ruminants, inhibits the enzyme ATPase, and is a blood-pressure-lowering agent.

DESCRIPTION OF THE DRAWINGS

The following infrared absorption spectra in chloroform are presented in the drawings:

FIG. 1 -- A-32887 (free acid)

FIG. 2 -- A-32887 mixed sodium-potassium salt

FIG. 3 -- A-32887 sodium salt

FIG. 4 -- A-32887 acetyl ester derivative (Na-K salt)

FIG. 5 -- A-32887 n-butyryl ester derivative (Na-K salt)

FIG. 6 -- A-32887 methyl ether derivative (Na salt)

DETAILED DESCRIPTION

The following paragraphs describe the properties of antibiotic A-32887.

A-32887 is a white, amorphous powder which melts at approximately 90° C. Elemental analysis of A-32887 indicates that it has the following approximate percentage composition (average): Carbon, 61.61%; Hydrogen, 8.56%; Oxygen, 28.63%. A-32887 has an approximate empirical formula of C₄₈₋₄₉ H₈₀₋₈₆ O₁₇₋₁₈ and a preferred empirical formula of C₄₈ H₈₂ O₁₈.

A-32887 has a molecular weight of about 946, as determined by mass spectrometry.

The infrared absorption spectrum of A-32887 (free acid) in chloroform is shown in FIG. 1 of the accompanying drawings. Significant absorption maxima occur at the following frequencies (cm⁻¹): 3540 (shoulder), 3420 (medium), 2945 (shoulder), 2905 (strong), 2860 (shoulder), 2805 (shoulder), 1710 (weak), 1450 (medium), 1370 (medium), 1350 (shoulder), 1300 (shoulder), 1275 (weak), 1170 (shoulder), 1155 (medium), 1100 (shoulder), 1085 (strong), 1060 (strong), 1010 (weak), 990 (weak), 980 (shoulder), 948 (medium), 890 (weak), and 850 (weak).

The ultraviolet spectrum of A-32887 shows no significant absorption.

The proton-magnetic-resonance spectrum of A-32887 indicates the presence of five methoxyl groups.

A-32887 (free acid) has the following specific rotation: [α]_(D) ²⁵ + 15.9° (c 1, CHCl₃).

A-32887 mixed sodium-potassium salt crystallizes from acetone-water and has a melting point of about 187°-190° C. Elemental analysis of A-32887 Na-K salt indicates that it has the following approximate percentage composition (average): Carbon, 60.14; Hydrogen, 8.11%; Oxygen, 29.64%; Sodium, 2.31%; Potassium, 0.46%.

The infrared absorption spectrum of A-32887 Na-K salt in chloroform is shown in FIG. 2 of the accompanying drawings. Significant absorption maxima are observed at the following frequencies (cm⁻¹): 3400 (shoulder), 3210 (medium), 2970 (strong), 2925 (strong), 2870 (weak), 2820 (weak), 1605 (medium), 1455 (medium), 1375 (shoulder), 1358 (medium), 1310 (weak), 1285 (weak), 1183 (medium), 1160 (medium), 1110 (shoulder), 1090 (strong), 1060 (strong), 1012 (weak), 980 (medium), 942 (medium), 910 (weak), 875 (shoulder), and 858 (weak).

A-32887 Na-K salt, crystallized from acetone-water, has the following characteristic X-ray powder diffraction pattern (CuNi, 1.5405 λ, d = interplanar spacing in angstroms):

    ______________________________________                                                             Relative                                                   d                   Intensity                                                  ______________________________________                                         12.53               50                                                         10.10               100                                                        9.5                 40                                                         9.05                40                                                         8.07                70                                                         7.16                100                                                        6.77                100                                                        6.48                70                                                         6.16                60                                                         5.58                50                                                         5.35                50                                                         5.10                30                                                         4.86                60                                                         4.70                50                                                         4.44                50                                                         4.25                50                                                         4.06                40                                                         3.80                30                                                         3.68                40                                                         3.58                30                                                         3.42                60                                                         3.22                10                                                         3.06                10                                                         2.97                10                                                         2.85                10                                                         2.76                02                                                         2.61                15                                                         2.49                05                                                         2.47                05                                                         2.44                15                                                         2.36                10                                                         2.29                10                                                         2.20                02                                                         2.12                02                                                         2.03                05                                                         1.96                05                                                         1.90                02                                                         1.81                02                                                         ______________________________________                                    

A-32887 Na-K salt has the following specific rotation:

    [α].sub.D.sup.25 + 9.6° (c 1, CHCl.sub.3).

Electrometric titration of A-32887 in 80% aqueous dimethylformamide indicates the presence of a titratable group with a pK_(a) value of 4.60.

A-32887 is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; is slightly soluble in non-polar organic solvents such as hexane and heptane; and is insoluble in water.

A-32887 is stable in aqueous solutions having a pH of from about 3 to about 11, but is unstable in solutions having a pH lower than about 3.

A-32887 sodium salt has a molecular weight of about 968, and A-32887 potassium salt has a molecular weight of about 984, both as determined by field-desorption (FD) mass spectrometry. The ion at m/e 969 (M + H) in the FD spectrum of A-32887 sodium salt was peak matched with a field-ionized mass standard. The found mass was 969.5373; the theoretical mass for C₄₈ H₈₂ O₁₈ Na is 969.5399. This finding supports a molecular formula of C₄₈ H₈₂ O₁₈ for A-32887 free acid.

In the following paper-chromatographic systems, using Bacillus subtilis ATCC 6633 bioautography for detection, A-32887 has these approximate R_(f) values:

    ______________________________________                                         Solvent System             R.sub.f Value                                       ______________________________________                                         Water saturated with methyl                                                    isobutyl ketone (MIBK)     0.58                                                Water:methanol:acetone                                                         (12:3:1). This solution                                                        is adjusted to pH 10.5     with                                                NH.sub.4 OH and then lowered                                                   to pH 7.5 with H.sub.3 PO.sub.4                                                                           0.32                                                Propanol:water (1:9)       0.85                                                Methanol:propanol:water (6:2:1).                                               Paper buffered with 0.75 M KH.sub.2 PO.sub.4,-pH 4.0.                                                     0.79                                                Methanol:0.05 M sodium citrate at                                              pH5.7 (7:3). Paper buffered with                                               0.05 M sodium citrate at pH 5.7.                                                                          0.85                                                Propanol:water (7:3)       0.91                                                ______________________________________                                    

In the following silica-gel TLC systems, using either vanillin spray reagent or Bacillus subtilis ATCC 6633 bioautography for detection, A-32887 has the these approximate R_(f) values:

    ______________________________________                                         Solvent System             R.sub.f Value                                       ______________________________________                                         Methanol                   0.80                                                Ethyl acetate:ethanol (1:4)                                                                               0.65                                                Ethyl acetate:chloroform (1:1)                                                                            0.36                                                Ethyl acetate:chloroform (6:1)                                                                            0.67                                                Benzene:ethyl acetate:methanol                                                 (6:4:0.2)                  0.58                                                ______________________________________                                    

A-32887 has an acid function capable of forming salts and ester derivatives and has at least one hydroxyl group capable of esterification. The C₂ -C₆ -acyl ester derivatives of A-32887 and the pharmaceutically-acceptable salts of these ester derivatives are also useful as antibiotics and as agents which increase feed-utilization efficiency.

The A-32887 acyl ester derivatives are typically prepared by reacting A-32887 with the corresponding C₂ -C₆ -acid anhydride or acid chloride at room temperature.

The following paragraphs describe characteristics of typical A-32887 acyl ester derivatives.

A-32887 acetyl ester derivative (Na-K salt) is a white amorphous powder which has a molecular weight of about 988 and a melting point of about 127°-129° C. A-32887 acetyl ester derivative has an approximate empirical formula of C₅₀₋₅₁ H₈₂₋₈₈ O₁₈₋₁₉.

The infrared absorption spectrum of A-32887 acetyl ester derivative (Na-K salt) in chloroform is shown in FIG. 4 of the accompanying drawings. Significant absorption maxima occur at the following frequencies (cm⁻¹): 3000, 2975, 2932, 2875, 2830, 1733, 1634, 1620, 1610, 1453, 1372, 1305, 1158, 1110, 1092, 1062, 1014, 996, 978, 948, 909, 893, and 853.

A-32887 n-butyryl ester derivative (Na-K salt) is a white amorphous powder which has a molecular weight of about 1016 and a melting point of about 59°-62° C. A-32887 n-butyryl ester derivative has an approximate empirical formula of C₅₂₋₅₃ H₈₆₋₉₂ O₁₈₋₁₉.

The infrared absorption spectrum of A-32887 n-butyryl ester derivative (Na-K salt) in chloroform is shown in FIG. 5 of the accompanying drawings. Significant absorption maxima occur at the following frequencies (cm⁻¹): 3000, 2968, 2932, 2875, 2830, 1724, 1630, 1620, 1610, 1592, 1452, 1372, 1355, 1179, 1155, 1110, 1090, 1060, 1012, 977, 946, and 892.

The C₂ -C₆ -acyl ester derivatives of A-32887 are soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; are slightly soluble in non-polar organic solvents such as hexane and heptane, and are insoluble in water.

A-32887 can be distinguished from its C₂ -C₆ acyl ester derivatives by TLC. For example, the acetyl and n-butyryl ester derivatives can be separated from A-32887 by silica-gel TLC using a benzene:ethyl acetate (1:1) solvent system. A sulfuric acid spray reagent can be used for detection. In this system A-32887 and its acetyl and n-butyryl ester derivatives have the following approximate R_(f) values:

    ______________________________________                                                               R.sub.f value                                            ______________________________________                                         A-32887 (Na-K)          0.47                                                   A-32887 acetyl ester                                                           derivative (Na-K)       0.40                                                   A-32887 n-butyryl ester                                                        derivative (Na-K)       0.64                                                   ______________________________________                                    

The A-32887 hydroxyl group can react with lower alkanols, lower-alkyl thiols, and glycols to form ether derivatives using procedures similar to those described for the preparation of A204I derivatives in U.S. Pat. No. 3,907,832. The A-32887 C₁ -C₄ -alkyl ether derivatives and their pharmaceutically-acceptable salts are especially useful as antibiotics and as agents which increase feed-utilization efficiency. Of the C₁ -C₄ -alkyl ether derivatives, the methyl ether derivative of A-32887 and its pharmaceutically-acceptable salts are preferred.

A-32887 methyl ether derivative has an approximate empirical formula of C₄₉₋₅₀ H₈₂₋₈₈ O₁₇₋₁₈. The sodium salt of A-32887 methyl ether derivative is a white crystalline (n-hexane:ethyl acetate) compound having a melting point of about 214-216° C.

The molecular weight of A-32887 methyl ether derivative sodium salt is about 982; the molecular weight of A-32887 methyl ether derivative free acid is about 960 (both as determined by FD mass spectrometry).

The infrared absorption spectrum of A-32887 methyl ether derivative (Na salt) in chloroform is shown in FIG. 6 of the accompanying drawings. Significant absorption maxima occur at the following frequencies (cm⁻¹): 3400 (broad), 2990, 2960, 2930, 2870, 2820, 1725, 1610, 1455, 1407, 1370, 1309, 1282, 1240, 1180, 1158, 1110, 1093, 1082, 1057, 1010, 980, 945, 900, 868, 858, 827, 802, 700, and 653.

The proton-magnetic-resonance spectrum of A-32887 methyl ether derivative (Na salt) indicates the presence of six methoxyl groups.

A-32887 methyl ether derivative (Na salt) has the following specific rotation: [α]_(D) ²⁵ - 5.1° (c 1, CHCl₃)

A-32887 methyl ether derivative (Na salt), crystallized from n-hexane:ethyl acetate, has the following characteristic X-ray powder diffraction pattern (CuNi, 1.5405 λ, d = interplanar spacing in angstroms):

    ______________________________________                                                             Relative                                                          d            Intensity                                                  ______________________________________                                                13.18        50                                                                12.01        50                                                                9.02         100                                                               8.26         100                                                               7.19         80                                                                6.46         50                                                                5.78         20                                                                5.46         20                                                                5.00         30                                                                4.24         20                                                                3.72         20                                                                3.36         05                                                         ______________________________________                                    

Electrometric titration of A-32887 methyl ether derivative (Na salt) in 80% aqueous dimethylformamide indicates the presence of a titratable group with a pK_(a) value of about 5.4.

A-32887 methyl ether derivative is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; is slightly soluble in non-polar organic solvents such as hexane and heptane; and is insoluble in water.

A-32887 methyl ether derivative (Na salt) can be separated from A-32887 (Na-K salt) by silica-gel TLC, using a benzene:ethyl acetate (1:1) solvent system and sulfuric acid spray reagent for detection. In this system, A-32887 and its methyl ether derivative have the following R_(f) values:

    ______________________________________                                                              R.sub.f value                                             ______________________________________                                         A-32887 (Na-K)         0.425                                                   A-32887 methyl ether   0.22                                                    derivative (Na)                                                                ______________________________________                                    

A-32887, the C₂ -C₆ -acyl ester derivatives of A-32887, and A-32887 methyl ether derivative are capable of forming salts. The pharmaceutically-acceptable alkali-metal, alkaline-earth-metal and amine salts of A-32887, the C₂ -C₆ -acyl ester derivatives of A-32887, and A-32887 methyl ether derivative are also part of this invention. "Pharmaceutically-acceptable" salts are those in which the toxicity of the compound as a whole toward warm-blooded animals is not increased relative to the non-salt form. Representative and suitable alkali-metal and alkaline-earth metal salts of A-32887 include the sodium, potassium, lithium, cesium, rubidium, barium, calcium, and magnesium salts. Suitable amine salts of A-32887 include the ammonium and the primary, secondary, and tertiary C₁ -C₄ -alkylammonium and hydroxy-C₂ -C₄ -alkylammonium salts. Illustrative amine salts include those formed by reaction of A-32887 with ammonium hydroxide, methylamine, sec-butylamine, isopropylamine, diethylamine, di-isopropylamine, ethanolamine, triethylamine, 3-amino-1-propanol and the like.

The alkali-metal and alkaline-earth-metal cationic salts of A-32887 are prepared according to procedures commonly used for the preparation of cationic salts. For example, the free-acid form of A-32887 is dissolved in a suitable solvent such as acetone; a solution containing the stoichiometric quantity of the desired inorganic base in aqueous acetone is added to this solution. The salt thus formed can be isolated by routine methods, such as filtration or evaporation of the solvent.

The salts formed with organic amines can be prepared in a similar manner. For example, the gaseous or liquid amine can be added to a solution of A-32887 in a suitable solvent such as acetone; the solvent and excess amine can be removed by evaporation.

It is well known in the veterinary pharmaceutical art that the the form of an antibiotic is not ordinarily of great significance when treating an animal with the antibiotic. In most cases, conditions within the animal change the drug to a form other than that in which it was administered. The salt form in which it may be administered is, therefore, not of great significance. The salt form may, however, be chosen for reasons of economy, convenience, and toxicity.

A-32887 is produced by culturing an A-32887-producing strain of Streptomyces albus under submerged aerobic conditions in a suitable culture medium until substantial antibiotic activity is produced. A-32887 is separated from the culture medium by the use of various isolation and purification procedures understood in the art.

The new microorganism useful for the preparation of antibiotic A-32887 was isolated from a soil sample collected in Curacao in Dutch Antilles. This organism is classified as a strain of Streptomyces albus (Rossi-Doria) Waksman and Henrici. This classification is based upon a comparison with the published description of the neotype strain ATCC 3004 [A. J. Lyons, Jr., and T. G. Pridham, J. Bacteriol. 83, 370-380 (1962)] and the Streptomyces albus strain IMRU 3005 [S. A. Waksman, "The Actinomycetes, Vol. II, Classification, Identification and Descriptions of Genera and Species," The Williams and Wilkins Co., Baltimore, 1961].

This classification is based on methods recommended by the International Streptomyces Project [E. B. Shirling and D. Gottlieb, "Methods for Characterization of Streptomyces Species," Intern. Bull. Systematic Bacteriol. 16, 313-340 (1966)] along with certain supplementary tests.

Color names were assigned according to the ISCC-NBS method (K. L. Kelly and D. B. Judd, "The ISCC-NBS Methods of Designating Colors and a Dictionary of Color Names," U.S. Dept. of Commerce Circ. 553, 1955, Washington, D.C.). Figures in parentheses refer to the Tresner and Backus color series [H. D. Tresner and S. J. Backus, "System of Color Wheels for Streptomycete Taxonomy," Appl. Microbiol. 11, 335-338 (1963)]. Color tab designations are underlined. Maerz and Paul color blocks (A. Maerz and M. R. Paul, "Dictionary of Color," McGraw-Hill Book Co., Inc., New York, N. Y., 1950) are enclosed in brackets.

Cell walls were prepared by a modified method of Heymann et al., "Structure of Streptococcal Cell Walls. I. Methylation Study of C-Polysaccharide," J. Biol. Chem. 238(2):502-509 (1963). Amino acids were determined by automatic amino-acid analysis by the modified method of Spackman et al., Anal. Chem. 30:1190-1206. Cultures were grown at 30° C. unless otherwise noted.

CHARACTERISTICS OF A-32887-PRODUCING STRAIN Morphology

Spiralled sporophores are produced. These are usually short. Although some chains have 3-10 spores per chain, there are usually more than 10 spores per chain. Spores are spherical to slightly oval and measure 0.78 μ × 0.56 μ with a range in size of 0.625 μ × 0.5 μ to 0.625 μ. The spores are smooth, as observed by electron micrographs. Morphologically, culture A-32887 could be confused with a verticillate type of morphology in which the branches are equidistant; however, the morphology actually is of a type in which the branching is very irregular.

                                      TABLE I                                      __________________________________________________________________________     CULTURAL CHARACTERISTICS ON VARIOUS MEDIA                                      Medium           Characteristics                                               __________________________________________________________________________     Czapek's-Solution Agar.                                                                         Abundant growth; reverse moderate yellow                                       [11K3]; abundant sporulation and aerial                                        mycelium; (W) a white; no soluble pigment.                    ISP Medium #2    Abundant growth; reverse moderate yellow                      (Yeast-Extract--Malt-Extract                                                                    [11K3]; abundant sporulation and aerial                       Agar)            mycelium; (W) a white; no soluble pigment.                    Tryptone--Yeast Agar.                                                                           Scant growth; reverse pale yellow [11C1];                                      scant sporulation and aerial mycelium; (W)                                     a white; no soluble pigment.                                  Nutrient Agar    Good growth; reverse pale yellow [11C1];                                       good aerial mycelium and sporulation; (W)                                      a white; no soluble pigment.                                  V-8 Juice--Dextrose Agar                                                                        Abundant growth; reverse moderate yellowish                                    brown [14E7]; abundant aerial mycelium and                                     sproulation; (W) a white; no soluble pigment.                 Glucose--Asparagine Agar                                                                        Good growth; reverse pale yellow [1102]; good                                  aerial mycelium and sporulation; (W) a white;                                  no soluble pigment.                                           Tomato-Paste--Oatmeal Agar                                                                      Abundant growth; reverse grayish yellow [12D3];                                abundant aerial mycelium and sporulatin; (W)                                   a white to (Y) 2 ba pale yellow; no soluble                                    pigment.                                                      Emerson's Agar   Abundant growth; reverse light yellow brown                                    [1217]; abundant aerial mycelium and sporula-                                  tion ; (W) a white to (GY) 2dc yellowish gray;                                 no soluble pigment.                                           ISP Medium #5    Scant growth; no color assignment due to poor                 (Glycerol--Asparagine Agar)                                                                     growth.                                                       Salts--Starch Agar                                                                              Good growth; reverse pale yellow [10B2]; good                                  aerial mycelium ad sporulation; (W) a white;                                   no soluble pigment.                                           ISP Medium #4    Scant-to-fair growth; reverse pale yellow                     (Inorganic-Salts--Starch Agar)                                                                  [9D2]; scant aerial mycelium and sporulation;                                  (W) a white; some clearing by area where inocu-                                lated; no soluble pigment. Growth not confluent                                but principally as isolated colonies.                         ISP Medium #3    Abundant growth; reverse pale yellow [10F2];                  (Oatmeal Agar)   abundant aerial mycelium and sporulation; (W)                                  a white; no soluble pigment.                                  Bennett's Modified(-COCl.sub.2) Agar                                                            Good-to-abundant growth; reverse pale yellow                                   [11B2]; abundant aerial mycelium and sporula-                                  lation; (W) a white to (GY) 2dc yellow-gray;                                   no soluble pigment.                                           Glycerol--Glycine Agar                                                                          Good growth; reverse pale yellow [11B2]; scant                                 aerial mycelium and sporulation; (W) a white;                                  no soluble pigment.                                           Tyrosine Agar    Fair-to-good growth; reverse grayish yellow                                    [12B3]; fair-to-good aerial mycelium and                                       sporulation; (W) a white; no soluble pigment.                 Calcium-Malate Agar                                                                             Good growth; reverse pale orange yellow; good                                  aerial mycelium and sporulation; (W) a white;                                  no soluble pigment.                                           __________________________________________________________________________

The organism was studied for selected physiologically properties in accordance with standard procedures. The properties observed and characteristics found are given in Table II:

                  TABLE II                                                         ______________________________________                                         Property Observed                                                                           Characteristics                                                   ______________________________________                                         Action on Skim Milk                                                                         No change after 14 days. A soft curd                                           is formed after 10 days incubation, but                                        milk is not cleared.                                              Starch Hydrolysis                                                                           Starch hydrolyzed                                                 Nitrate Reduction                                                                           Negative                                                          Gelatin Liquefaction                                                                        None at 14 days                                                   Melanin Pigment                                                                Production on:                                                                 1. Tryptone--Yeast-                                                              Extract Broth.                                                                            Negative                                                          2. Peptone--Yeast-                                                               Extract--Iron Agar.                                                                       Negative                                                          3. Tyrosine Agar                                                                            Negative                                                          Growth on:                                                                       Carrot slice                                                                              Scant vegetative growth.                                            Potato slice                                                                              Abundant growth and sporulation; aerial                                        and spores off-white to brownish gray.                            Temperature                                                                    No growth.ts 20°                                                        (Bennett's-agar slants;                                                        Good growth; white aerial-reverse                                              incubated 9 days)                                                                           yellow brown; no soluble pigment.                                 Good growth; white aerial-reverse                                                           yellow brown; no soluble pigment.                                 Good growth; white aerial-reverse                                                           yellow brown; no soluble pigment.                                 Fair growth; scant white aerial-                                                            reverse color darker than at 25°, 30°               brown; brown soluble pigment.                                                  No growth.   49°                                                        No growth.   55°                                                        ______________________________________                                    

The response to the culture to varying levels of sodium chloride, using Bennett's modified agar, is summarized in Table III.

                  TABLE III                                                        ______________________________________                                         Percent NaCl                                                                   in Medium   Characteristics                                                    ______________________________________                                         1           Good-to-abundant growth; aerial                                                mycelium and spores; white aerial.                                 2           Abundant growth; abundant aerial                                               mycelium and spores; white aerial.                                 3           Abundant growth; abundant aerial                                               mycelium and spores; white aerial.                                 4           Abundant growth; abundant aerial                                               mycelium and spores; white aerial.                                 6           Good-to-abundant growth; abundant                                              aerial mycelium and spores; white                                              aerial.                                                            8           Good veg. growth; no aerial                                                    mycelium.                                                          10          Fair-to-good veg. growth; no                                                   aerial mycelium.                                                   12          Scant growth; no aerial myce-                                                  lium.                                                              14          Scant-to-no growth; no aerial                                                  mycelium.                                                          ______________________________________                                    

The results of carbon-utilization tests carried out with the organism are set forth in Table IV. The following symbols are used:

+ = Positive utilization

(+) = Probable utilization

(-) = Questionable utilization

- = No utilization

                  TABLE IV                                                         ______________________________________                                         Carbon Source         Response                                                 ______________________________________                                         L-arabinose           +                                                        D-ribose              (+)                                                      D-xylose              (+)                                                      D-galactose           (+)                                                      D-glucose             (+)                                                      D-mannose             (+)                                                      D-fructose            (+)                                                      L-sorbose             -                                                        cellobiose            (+)                                                      lactose               (+)                                                      maltose               +                                                        melibiose             (-)                                                      sucrose               (+)                                                      turanose              (-) to -                                                 trehalose             +                                                        melezitose            (-)                                                      raffinose             (-) to -                                                 dextrin               +                                                        salicin               -                                                        starch (soluble)      +                                                        fucose                (+)                                                      rhamnose              +                                                        glucosamine           (+)                                                      α-methylglucoside                                                                              -                                                        α-methylxyloside                                                                               -                                                        adonitol              (+)                                                      dulcitol              (+) to -                                                 i-erythritol          (+)                                                      glycerol              (+)                                                      i-inositol            +                                                        mannitol              +                                                        sorbitol              +                                                        Carbon (Negative                                                               Control)              -                                                        ______________________________________                                    

Cell Wall Studies

Using hydrolyzed whole cells of the organism, the presence of certain diagnostic sugars was determined. Isolated cell walls were used to determine the isomers of diaminopimelic acid and the amino-acid content. The results of these cell-wall studies are set forth below:

    ______________________________________                                         Test              Result Observed                                              ______________________________________                                         Isomers of diamino-                                                            pimelic acid      LL-isomer                                                    Diagnostic sugars No characteristic                                                              pattern                                                      Amino-acid content                                                                               Major amounts of                                                               glutamic acid, glycine,                                                        alanine, and tyrosine.                                       ______________________________________                                    

Certain characteristics of the A-32887-producing S. albus strain differ from those in the published description of S. albus (Rossi-Doria) Waksman and Henrici, supra. A comparison of the characteristics of the A-32887-producing strain (NRRL 11109) with those in the published description is given in Table V; differing characteristics are highlighted by an asterisk.

                                      TABLE V                                      __________________________________________________________________________                    Reaction of A-32887                                                                         Published Description of                           Medium; Condition                                                                             S. albus NRRL 11109                                                                         Streptomyces albus                                 __________________________________________________________________________     ISP Medium #2  White aerial; moderate                                                                      White or yellow aerial; yellow                     (Yeast--Malt-Extract Agar)                                                                    yellow reverse                                                                              brown reverse                                      ISP Medium #3  White aerial; pale yel-                                                                     White aerial; yellow brown reverse                 (Oatmeal Agar) low reverse                                                     ISP Medium #4  White aerial; pale yel-                                                                     White aerial; yellow brown reverse                 (Inorganic-Salts--Starch Agar)                                                                low reverse                                                     ISP Medium #5  Scant growth; no color                                                                      White or yellow aerial; yellow                     (Glycerol Asparagine Agar)                                                                    assignment   brown reverse                                      Reacton to various levels of                                                                  Tolerates levels of up                                                                      Tolerates levels greater than 13%.                 NaCl           to 12%. No growth at                                                                        No growth at 15%                                                  14%.                                                            Temperature Requirements                                                                      Optimum growth at 25° -                                                              Optimum growth at 25° - 44° C                       43° C                                                    Reaction to the following                                                      carbon sources:                                                                D-Glucose      (+)          +                                                  D-Mannitol     +            +                                                  D-Galactose    (+)          +                                                  L-Arabinose*   +            -                                                  Rhamnose*      +            -                                                  D-Xylose       (+)          +                                                  i-Inositol*    +            -                                                  D-Fructose     (+)          ±                                               Salicin*       -            +                                                  Raffinose      (-)          -                                                  Whole-cell Hydrolysates                                                                       LL-diaminopimelic acid                                                                      LL-diaminopimelic acid                             Nitrate Reduction*                                                                            Negative     Positive                                           Gelatin Liquefaction*                                                                         None at 14 days                                                                             Strong liquefaction                                Melanin-pigment Production                                                                    Negative     Negative                                           Action on Skim Milk*                                                                          No change after 14 days.                                                                    Rapid peptonization                                               Soft curd is formed after                                                      10 days, but milk is not                                                       cleared.                                                        Morphology     Spiralled    Spiralled                                          Spore ornamentation                                                                           Smooth       Smooth                                             __________________________________________________________________________

The Streptomyces albus culture useful for the production of antibiotic A-32887 has been deposited and made a part of the stock culture collection of the Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill. 61604, from which it is available to the public under the number NRRL 11109.

As is the case with other organisms, the characteristics of the A-32887-producing culture, Streptomyces albus NRRL 11109, are subject to variation. For example, artificial variants and mutants of the NRRL 11109 strain may be obtained by treatment with various known mutagens such as ultraviolet rays, X-rays, high-frequency waves, radioactive rays, and chemicals. All natural and artificial variants and mutants which have the essential identifying characteristics of Streptomyces albus and produce A-32887 may be used in this invention. "Essential identifying characteristics" are those characteristics which are sufficient to classify an organism as Streptomyces albus NRRL 11109. One of these characteristics, of course, is the ability of the organism to produce A-32887. It will be understood by those skilled in the art that certain non-critical differences between the characteristics exhibited by a given organism and those identifying a reference organism can exist without affecting the classification of both such organisms as belonging to the same genus, species and strain.

The culture medium employed to grow Streptomyces albus NRRL 11109 can be any one of a number of media. For economy in production, optimal yield, and ease of product isolation, however, certain culture media are preferred. Thus, for example, a preferred carbohydrate source in large-scale fermentation is glucose, although dextrin, starch, maltose, and the like can also be used. A preferred nitrogen source is meat peptone, although other peptones, enzyme-hydrolyzed casein, soybean meal, amino acids and the like are also useful. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, potassium, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, and like ions.

Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other substituents of the medium in amounts sufficient to meet the growth requirements of the organism. It may be necessary to add small amounts (i.e. 0.2 ml/l.) of an antifoam agent such as polypropylene glycol (M.W. about 2000) to large-scale fermentation media if foaming becomes a problem.

For production of substantial quantities of antibiotic A-32887, submerged aerobic fermentation in tanks is preferred. Small quantities of antibiotic A-32887 may be obtained by shake-flask culture. Because of the time lag in antibiotic production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the vegetative inoculum can be the same as that employed for larger fermentations, but other media can also be employed.

The A-32887-producing organism can be grown at temperatures between about 22° and about 45° C. Optimum A-32887 production appears to occur at temperatures of about 30° C.

As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium. For efficient production of antibiotic A-32887 the volume of air employed in tank productions is preferably about 0.25-0.5 volume of air per volume of culture medium per minute (V/V/M).

Production of antibiotic A-32887 can be followed during the fermentation by testing samples of the broth or of extracts of the mycelial solids for antibiotic activity against organisms known to be sensitive to this antibiotic. One assay organism useful in testing this antibiotic is Bacillus subtilis ATCC 6633. The bioassay is conveniently performed by paper-disc assay on agar plates.

Following its production under submerged aerobic fermentation conditions, antibiotic A-32887 can be recovered from the fermentation medium by methods employed in the fermentation art. Although the antibiotic activity produced during fermentation of the A-32887-producing organism occurs in both the broth and in the mycelial mass, the major part of the activity is in the filtered broth. Maximum recovery of antibiotic A-32887 is accomplished, therefore, by an initial filtration to separate the broth from the mycelial mass. The filtered broth can then be further purified to give antibiotic A-32887. A variety of techniques may be used in this purification. A preferred technique for purification of the filtered broth involves adjusting the broth to about pH 9 and extracting with a suitable solvent such as ethyl acetate. The extracting solvent can then be evaporated under vacuum to give partially-purified antibiotic A-32887. Further purification of A-32887 involves the use of chromatography. A preferred adsorbent for this purification is silica gel.

Alternatively, the culture solids, including medium constituents and mycelium can be used without extraction of separation, but preferably after removal of water, as a source of antibiotic A-32887. For example, after production of A-32887 antibiotic activity, the whole fermentation broth or the broth filtrate can be dried by lyophilization, by drum-drying, or by azeotropic distillation and drying. The dried whole broth or dried broth filtrate can then be mixed directly into feed premix.

Antibiotic A-32887 inhibits the growth of pathogenic bacteria, especially gram-positive bacteria. Table VI summarizes the minimal inhibitory concentrations (MIC), as measured by standard agar-dilution assays, at which A-32887 (Na-K salt) inhibits certain bacteria.

                  TABLE VI                                                         ______________________________________                                         Test Organism          MIC (mcg/ml)                                            ______________________________________                                         Staphylococcus aureus 3055                                                                            6.25                                                    Streptococcus faecalis 6.25                                                    Staphylococcus sp.     1.56                                                    Streptococcus sp.      3.12                                                    Pasteurella multocida (bovine)                                                                        50.00                                                   Pseudomonas sp.        3.12                                                    ______________________________________                                    

The activity of A-32887 (Na-K salt) against illustrative bacteria, as measured by the conventional disc-diffusion method, is summarized in Table VI.

                  TABLE VII                                                        ______________________________________                                                                     Zone of                                            Test Organism      mcg/disc Inhibition (mm)                                    ______________________________________                                         Staphylococcus aureus 3055                                                                        300      15.6                                               Staphylococcus aureus 3055                                                                         30      12.0                                               Staphylococcus aureus 3074*                                                                       300      16.0                                               Staphylococcus aureus 3074*                                                                        30      12.7                                               Staphylococcus aureus 3130**                                                                      300      15.2                                               Staphylococcus aureus 3130**                                                                       30      15.0                                               Streptococcus pyogenes (Group A)                                                                  300      17.5                                               Streptococcus pyogenes (Group A)                                                                   30      14.0                                               Streptococcus sp. (Group D)                                                                       300      14.7                                               Streptococcus sp. (Group D)                                                                        30      13.7                                               Diplococcus pneumoniae                                                                            300      18.0                                               Diplococcus pneumoniae                                                                             30      16.0                                               ______________________________________                                          *Penicillin G-resistant                                                        **Methicillin-resistant                                                  

The antimicrobial activity of two typical A-32887 acyl ester derivatives is compared with that of A-32887 (each as Na-K salts) in Table VIII. Activity against illustrative bacteria is measured by the conventional disc-diffusion method. In addition, the results of a conventional paper-disc agar-diffusion assay system (Plate Assay) against Bacillus subtilis ATCC 6633 are reported. The activity in this test is quantitated and uses a dried-broth reference standard which is assigned an arbitrary potency of 100 units/ml. The samples were assayed at 1 mg/ml.

                                      TABLE VIII                                   __________________________________________________________________________               Plate Assay                                                                          Staphylococcus                                                                         Bacillus                                                                            Micrococcus                                                                           Bacillus                                   Compound  units/ml                                                                             aureus  subtilis                                                                            lutea  subtilis*                                  __________________________________________________________________________     A-32887   1350  16      18   16     30                                         A-32887 Acetyl                                                                 Ester Derivative                                                                          238  trace   12   trace  22                                         A-32887 n-Butyryl                                                              Ester Derivative                                                                         1109  14      16   12     27                                         __________________________________________________________________________      *Minimal media                                                           

In one important aspect, the A-32887 compounds inhibit the growth of anaerobic bacteria. Table IX summarizes the MIC's at which A-32887 (Na-K salt) inhibits various anaerobic bacteria, as determined by standard agar-dilution assay. End points were read after 24-hour incubation.

                  TABLE IX                                                         ______________________________________                                         Test Organism          MIC (mcg/ml)                                            ______________________________________                                         Actinomyces israelii   ≦0.5                                             Clostridium perfringens                                                                               ≦0.5                                             Clostridium septicum   ≦0.5                                             Eubacterium aerofaciens                                                                               ≦0.5                                             Peptococcus asaccharolyticus                                                                          ≦0.5                                             Peptococcus prevoti    ≦0.5                                             Peptostreptococcus anaerobius                                                                         ≦0.5                                             Peptostreptococcus intermedius                                                                        1.0                                                     Propionibacterium acnes                                                                               ≦0.5                                             Bacteriodes fragilis ssp                                                       fragilis 111           2                                                       Bacteriodes fragilis ssp                                                       fragilis 1877          2                                                       Bacteriodes fragilis ssp                                                       fragilis 1936B         2                                                       Bacteriodes fragilis ssp                                                       thetaiotaomicron       2                                                       Bacteriodes melaninogenicus                                                    1856/28                32                                                      Bacteriodes melaninogenicus                                                    2736                   2                                                       Bacteriodes vulgatis   4                                                       Bacteriodes corrodens  2                                                       Fusobacterium symbiosum                                                                               32                                                      Fusobacterium necrophorum                                                                             32                                                      ______________________________________                                    

The activity of the A-32887 compounds against anaerobic bacteria, especially against Clostridium perfringens, suggests that the A-32887 compounds would be beneficial in the treatment or prevention of enteritis in chickens, swine, cattle, sheep, and goats and in the treatment or prevention of enterotoxemia in ruminants.

Activity against mycoplasma is another useful aspect of the antimicrobial activity of the A-32887 compounds. Mycoplasma species, also known as pleuropneumonia-like (PPLO) organisms, are pathogenic to man and various animals. Anti-mycoplasma agents are especially needed by the poultry industry. The MIC's of A-32887 (Na-K salt) against typical mycoplasma species, as determined by in vitro broth-dilution studies, are summarized in Table X:

                  TABLE X                                                          ______________________________________                                         Test Organism        MIC (mcg/ml)                                              ______________________________________                                         Mycoplasma gallisepticum                                                                            12.5                                                      Mycoplasma synoviae  3.12                                                      Mycoplasma hyorhinis 25.0                                                      Mycoplasma hyopneumoniae                                                                            6.25                                                      ______________________________________                                    

The A-32887 compounds are also antiviral agents. For example, A-32887 is active against type B influenza virus (Maryland, dog kidney), Transmissible Gastroenteritis virus and Infectious Canine Hepatitis virus, as demonstrated by in vitro plaque suppression tests, similar to that described by Siminoff, Applied Microbiology 9 [1], 66-72 (1961).

The acute toxicity of A-32887 (Na-K salt), when administered intraperitoneally to mice and expressed as LD₅₀, is 37.5 mg/kg × 1.

A most important property of the A-32887 compounds is their anticoccidial activity. For example, feeding experiments show that A-32887 (Na-K salt), when present in the feed of young chickens at levels as low as 40 ppm, decreases mortality and the number of lesions in chicks which have been challenged with coccidia. Tables XI through XIII summarize the results of tests with A-32887 (Na-K salt) in chicks challenged with various Eimeria species.

                                      TABLE XI                                     __________________________________________________________________________     ACTIVITY OF A-32887 AGAINST E. tenella AND E. maxima.sup.1                                         Average        Average Lesion Score                        Treatment .sup.2,3                                                                        PPM                                                                               Mortality.sup.4                                                                      Weight Gain (g).sup.5                                                                   Feed/Gain.sup.6                                                                      Total Intestinal                                                                       Cecal                               __________________________________________________________________________     Normal Controls                                                                            0 0     195      1.65  0       0                                   Infected Controls                                                                          0 40     66      --    10.8    3.7                                 A-32887 (Na-K salt)                                                                       100                                                                               0     159      1.87  1.6     0                                   A-32887 (Na-K salt)                                                                       80 0     182      1.83  3.0     0.3                                 A-32887 (Na-K salt)                                                                       60 0     155      1.83  5.9     0.6                                 A-32887 (Na-K salt)                                                                       40 0     176      1.89  4.9     1.6                                 __________________________________________________________________________      .sup.1 70,000 oocysts/bird of each of E. tenella and E. maxima                 .sup.2 Infected 48 hours postmedication; terminated seven days                 postinoculation                                                                .sup.3 Four replicates; five birds/replicate                                   .sup.4 Due to coccidiosis                                                      .sup.5 Per survivor                                                            .sup.6 Pens without deaths only                                          

                  TABLE XII                                                        ______________________________________                                         ACTIVITY OF A-32887 AGAINST                                                    E. tenella AND E. acervulina                                                             Per-           Average                                                         cent  Percent  Lesion Score                                                                              Oocysts                                                    Morta-  Weight Intest-                                                                              Ce-  Per Bird                             Treatment                                                                               ppm    lity.sup.3                                                                             Gain.sup.4                                                                            inal  cal  (1×10.sup.6).sup.5             ______________________________________                                         Infected --     12.5    63     1.2   3.6  29.37                                Controls                                                                       A-32887  121    0       73     0.3   0    0.4                                  (Na-K salt)                                                                    A-32887  100    0       85     0.6   0.1  1.28                                 (Na-K salt)                                                                    A-32887   77    0       90     0.7   0    5.48                                 (Na-K salt)                                                                    A-32887   62    0       97     0.9   0.1  16.40                                (Na-K salt)                                                                    ______________________________________                                          .sup.1 6 replicates; 5 birds each                                              .sup.2 Infection inoculum 48 hours post-medication onset                       .sup.3 Due to coccidiosis                                                      .sup.4 Normal controls = 100%                                                  .sup.5 Days 7-9                                                          

                                      TABLE XIII                                   __________________________________________________________________________     ACTIVITY OF A-32887 AGAINST E. tenella, E. maxima and E. acervulina                           Percent                                                                               Percent        Average Lesion Score                                                                        Oocysts/Bird.sup.6           Treatment.sup.1,2                                                                         ppm Mortality.sup.3                                                                       Weight Gain.sup.4                                                                      Feed/Gain.sup.5                                                                       Intestinal                                                                            Cecal 1×10.sup.6             __________________________________________________________________________     Normal Controls                                                                           --  0      100     1.46   --     --    --                           Infected Controls                                                                         --  32.5   40      --     1.2    3.6   63.54                        A-32887 (Na-K salt)                                                                       100 0      93      1.50   0      0.1    6.49                        A-32887 (Na-K salt)                                                                       75  0      90      1.49   0.9    2.0   24.35                        A-32887 (Na-K salt)                                                                       50  0      79      1.67   1.8    3.8   35.78                        A-32887 (Na-K salt)                                                                       25  35     57      --     1.1    3.9   64.83                        __________________________________________________________________________      .sup.1 7 days post-inoculation; infection inoculum 24 hrs. post-medicatio      onset                                                                          .sup.2 Six replicates; 5 birds/each                                            .sup.3 Due to coccidiosis                                                      .sup.4 Per survivor                                                            .sup.5 Pens without deaths, only                                               .sup.6 Total days 5, 6 and 7 post-inoculation                            

For the prevention or treatment of coccidiosis in poultry, a non-toxic anticoccidial amount of an A-32887 compound is administered to birds, preferably orally on a daily basis. The A-32887 compound can be supplied in many ways, but is is most conveniently supplied with a pharmaceutically-acceptable carrier, preferably the feed ingested by the birds. Although a variety of factors must be considered in determining an appropriate concentration of A-32887 compound, the rates of administration are generally in the range of about 30 to about 180 ppm in the feed and are preferably in the range of about 50 to about 120 ppm of feed ration.

This invention further relates to feed compositions adapted to protect poultry from coccidiosis and containing from about 45 to about 110 pounds of A-32887 compound per ton of poultry feed.

Another important property of the A-32887 compounds is their ability to improve feed-utilization efficiency in ruminants which have a developed rumen function. It is known that the efficiency of carbohydrate utilization in ruminants is increased by treatments which stimulate the animals' rumen flora to produce propionate compounds rather than acetate or butyrate compounds (for a more complete discussion see Church et al. in "Digestive Physiology and Nutrition of Ruminants", Vol. 2, 1971, pp 622 and 625).

The efficiency of feed use can be monitored by observing the production and concentration of propionate compounds in the rumen using the method described by Arthur P. Raun in U.S. Pat. No. 3,839,557 (see especially Example 6).

Table XIV shows the ratio of volatile-fatty-acid (VFA) concentrations in A-32887-treated flasks to concentrations in control flasks in this test.

                                      TABLE XIV                                    __________________________________________________________________________                               Ratio of Treated to Control                                             Molar %                                                                               Molar %                                                                               Molar %                                                                               Total VFA                              Compound  Dose(mcg/ml)                                                                            Propionate                                                                            Acetate                                                                               Butyrate                                                                              mM/l.                                  __________________________________________________________________________     A-32887 (Na-K)                                                                           1        1.4191*                                                                               0.5744 1.1116 0.7498                                 A-32887 (Na-K)                                                                           10       1.2919*                                                                               0.8857 0.7012 1.0086                                 A-32887 Methyl                                                                 Ether Derivative                                                               (Na-K)    1        1.1219*                                                                               0.9451 0.8166 1.1286                                 A-32887 Methyl                                                                 Ether Derivative                                                               (Na-K)    5        1.2638*                                                                               0.8997 0.7404 1.0693                                 __________________________________________________________________________      *Statistically significant (P<0.01) by the two-tailed LSD test (R.G.D.         Steel and J.H. Torrie, "Priciples and Procedures of Statistics",               McGraw-Hill, New York, N.Y., 1960, p. 106)                               

Carbohydrate-utilization efficiency is further measured by in vivo tests performed in animals which have had a fistula installed in the rumen, making it possible to withdraw specimens of the rumen contents. The procedure used in testing cattle in this manner is also described in Raun's U.S. Pat. No. 3,839,557 (see Example 9). Table XV summarizes the results of such a test with A-32887 (Na-K salt) wherein five feed-lot cattle weighing approximately 425 kg. were in each group and the mean percent increases in ruminal propionate concentration were averaged over four analyses in a 24-day treatment period.

                  TABLE XV                                                         ______________________________________                                                    Molar %   Molar %  Molar %                                                                               Total VFA                                 Treatment  Propionate                                                                               Acetate  Butyrate                                                                              (mM/l.)                                   ______________________________________                                         Control    17.4      74.5     8.1    79.9                                      A-32887 (Na-K                                                                             27.1*     63.3*    9.6    79.6                                      salt) 30 g/ton                                                                 ______________________________________                                          *Significantly different (P<0.01) from control by the two-tailed LSD test                                                                               

Table XVI summarizes the results of a similar test in sheep with A-32887 (Na-K salt) wherein four sheep were in each group and the mean percent increases in ruminal propionate concentration were averaged over four analyses in a 19-day treatment period.

                  TABLE XVI                                                        ______________________________________                                                               Molar %                                                  Treatment             Propionate                                               ______________________________________                                         Control               26.5                                                     A-32887 (Na-K salt)   29.5                                                     15 g/ton                                                                       ______________________________________                                    

The A-32887 compounds are typically effective in increasing propionates and, thereby, the efficiency of feed-utilization efficiency when administered to ruminants orally at rates of from about 0.07 mg/kg/day to about 4.0 mg/kg/day. Most beneficial results are achieved at rates of from about 0.2 mg/kg/day to about 2.0 mg/kg/day.

A preferred method of administration is to mix the A-32887 compound with the animals' feed; however, it can be administered in other ways, for example, tablets, drenches, sustained-release boluses, or capsules. Formulation of these various dosage forms can be accomplished by methods well known in the veterinary pharmaceutical art. Each individual dosage unit should contain a quantity of A-32887 compound directly related to the proper daily dose for the animal to be treated.

This invention further relates to feed compositions adapted to fatten cattle comprising cattle feed and from 1 to 25 grams per ton of an A-32887 compound.

In another aspect the A-32887 compounds are useful in the prevention and control of swine dysentery. A preferred method of administration to swine is by incorporation of an appropriate amount of an A-32887 compound into the feed ration. The results of tests with A-32887 (Na-K salt) when administered to pigs infected with acute swine dysentery are reported in Table XVII. In this test, groups of four pigs were challenged orally with 5.0 ml of a colon-content/tissue suspension prepared from pigs suffering from acute swine dysentery. Treated pigs were challenged 24 hours after initiating feed treatment. The test was carried out over a period of 26 days, observing pigs daily, and weighing them weekly and on day 26.

                                      TABLE XVII                                   __________________________________________________________________________                                            No. with Colon                                     Final Average Wt.                                                                            No. Died per                                                                           Diarrhea                                                                             Lesions/No. in                          Treatment  per Pig (lbs) No. in Group                                                                           Index*                                                                               Group                                   __________________________________________________________________________     A-32887 (Na-K salt)                                                            100 g/ton  30.3          0/4     22    2/4                                     Infected Non-                                                                  medicated Controls                                                                        14.3          3/4     53    4/4                                     Infected Non-                                                                  medicated Controls                                                                        14.8          2/4     52    4/4                                     __________________________________________________________________________      *Fecal material for each group was rated daily with 0= normal, 1= slight       blood or mucus, 2= moderate blood or mucus, 3= marked blood or mucus.          Index is the total score per treatment for 25 days.                      

The A-32887 compounds are also useful in the treatment of certain plant diseases. For example, A-32887 (Na-K salt), when applied at 400 ppm as a spray, inhibits powdery mildew disease in bean plants.

In another aspect, the A-32887 compounds are useful as insecticides. For example, A-32887 (Na-K salt) is active against insects such as Mexican bean beetle, Southern armyworm and housefly when applied at a rate of 1000 ppm.

Antibiotic A-32887 exhibits ion-binding and ion-transport properties and is, therefore, an ionophore (ion-bearer) (see B. C. Pressman, Alkali metal chelators -- the ionophores, in "Inorganic Biochemistry," Volume 1, G. L. Eichhorn, Elsevier, 1973). A-32887 can be used when the selective removal of particular cations is desired. Examples of such uses include the removal and recovery of silver ions from solutions in photography, the removal of toxic cations from industrial waste streams before such streams are discharged to the environment, and desalinization of sea water. A-32887 can be used as one component of an ion-specific electrode (O. Kedem, et al., U.S. Pat. No. 3,753,887, Aug. 21, 1973, Alkali metal specific measuring electrode). A-32887 alters the cation permeability of both natural and artificial membranes. A-32887 can be used, therefore, as a component in a membrane used for the selective transport of cations against a concentration gradient. One potential application of this property is in recovery of heavy and precious metals on a commercial basis [see E. L. Cussler, D. F. Evans, and Sister M. A. Matesick, Science 172, 377 (1971)].

In yet another aspect, the A-32887 compounds are active as inhibitors of the enzyme ATPase. ATPase, an alkali-metal-sensitive enzyme found in cell membranes, is involved in the energy necessary for active transport. "Active transport" refers to the energy-requiring series of operations whereby intracellular and extracellular fluids maintain their compositions. Inhibitors of ATPase reduce the energy required for active transport. A-32887 (Na-K salt) has been shown to inhibit ATPase in in vitro tests using NaCl.

The A-32887 compounds are potential cardiotonic agents. In tests using guinea pig left atria, A-32887 (Na-K salt) increased cardiac contractility. Response to this test is expressed as a percentage of the maximal force that could be elicited by a challenge dose of norepinephrine. A-32887 (Na-K salt), at a 10⁻⁵ molar concentration, increased cardiac contractility by 28.0 ± 7.1 percent.

In order to illustrate more fully the operation of this invention, the following examples are provided:

EXAMPLE 1 A. Shake-flask Fermentation of A-32887

A lyophilized pellet of Streptomyces albus NRRL 11109 was dissolved in 1-2 ml of sterilized water. This solution was used to inoculate an agar slant having the following composition:

    ______________________________________                                         Ingredient             Amount                                                  ______________________________________                                         Agar                   20 g                                                    Dextrin                10 g                                                    Yeast extract          1 g                                                     Beef extract           1 g                                                     Enzymatic hydrolysate                                                          of casein*             2 g                                                     CoCl.sub.2 . 6H.sub.2 O                                                                               0.01 g                                                  Deionized water        q.s. 1 liter                                            ______________________________________                                          NaOH was added to raise the pH of the medium from about 6.2 to about 7.0,      before sterilizing; pH after sterilization about 6.9.                          *NZ Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.                   

The inoculated slant was incubated at 30° C. for about 7 days. The mature slant culture was scraped with a sterile pipette or loop to loosen the spores. About one-fourth of the loosened spores were used to inoculate 50 ml of a vegetative medium having the following composition:

    ______________________________________                                         Ingredient            Amount                                                   ______________________________________                                         Glucose               15 g                                                     Soybean meal          15 g                                                     Corn steep liquor     10 g                                                     NaCl                  5 g                                                      CaCO.sub.3            2 g                                                      Cold tap water        q.s. 1 liter                                             ______________________________________                                          The pH of this medium was adjusted from approximately 5.8 to about 6.5 by      the addition of NaOH; post-sterilization pH about 6.5.                   

The inoculated vegetative medium was incubated in a 250-ml Erlenmeyer flask at 30° C. for about 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

This incubated vegetative medium (0.5-2.5 ml; 1-5%) was used to inoculate 50 ml of a production medium having the following composition:

    ______________________________________                                         Ingredient            Amount (g/l.)                                            ______________________________________                                         Glucose               25.0                                                     Starch                10.0                                                     Peptone*              10.0                                                     Enzymatic-hydrolysate of                                                       casein**              4.0                                                      Blackstrap molasses   5.0                                                      MgSO.sub.4.7H.sub.2 O 0.5                                                      CaCO.sub.3            2.0                                                      Czapek's mineral stock***                                                      Deionized water       q.s. 1 liter                                             ______________________________________                                          *Wilson's Peptone 159, Wilsons' Protein Technology                             **NZ Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.                        ***Czapek's mineral stock has the following composition: 100 g KCl; 100 g      MgSO.sub.4.7H.sub.2 O; 2 g FeSO.sub.4.7H.sub.2 O; q.s. to 1 liter with         deionized water                                                          

The inoculated production medium was incubated in a 250-ml Erlenmeyer flask at 30° C. for about 2 to 3 days on a shaker rotating through an arc two inches in diameter at 250 RPM.

B. Tank Fermentation of A-32887

In order to provide a larger volume of inoculum, 20 ml of incubated vegetative medium, prepared as described in Section A, was used to inoculate 400 ml of a second-stage vegetative-growth medium having the same composition as that of the vegetative medium. This second-stage vegetative medium was incubated in a 2-liter flask for about 24 hours at 30° C. on a shaker rotating through an arc two inches in diameter at 250 RPM.

Incubated second-stage medium (800 ml) thus prepared was used to inoculate 100 liters of sterile production medium, prepared as described in Section A. The inoculated production medium was allowed to ferment in a 165-liter fermentation tank for 3 to 4 days at a temperature of 30° C. The fermentation medium was aerated with sterile air at the rate of 0.25 V/V/M and was stirred with conventional agitators at 250 RPM.

EXAMPLE 2 Separation of A-32887

Whole fermentation broth (925 l.), obtained by the method described in Example 1, was adjusted to pH 8.5 by the addition of NaOH, stirred for 45 minutes, and filtered with a filter aid (Hyflo Super-cel, a diatomaceous earth, Johns-Manville Products Corp.). The filtered cake was washed with water, and the water wash was added to the filtered broth. The filtered-broth solution was then extracted twice with ethyl acetate (2/3 volumes). The ethyl acetate extracts were combined and concentrated under vacuum to give an oily residue. The residue was dissolved in benzene (4 l.); the benzene solution was filtered; and the filtrate was applied to a 9.5- × 162-cm silica-gel column (Grace, grade 62), prepared in benzene. After washing the column with benzene (24 l.), the eluting solvent was changed to benzene:ethyl acetate (3:2), collecting 37 liters consisting of fractions of 1 liter each. Elution was monitored by silica-gel thin-layer chromatography, using a benzene:ethyl acetate (1:1) solvent system and Bacillus subtilis bioautography for detection. The active fractions which contained A-32887 (11 l.) were combined and evaporated to dryness under vacuum. In order to remove color and other impurities, the residue thus obtained was dissolved in chloroform and chromatographed on a 2.2- × 40-cm column of carbon (Pittsburgh 12 × 40), prepared in chloroform. The column was washed with chloroform (3 l.); the chloroform eluate was concentrated to dryness under vacuum. The residue thus obtained was dissolved in diethyl ether (200 ml). The resulting solution was evaporated slowly under vacuum to give a thick syrup. The syrup was slowly warmed, and n-hexane (500 ml) was added with stirring. This solution was allowed to stand at room temperature until the A-32887 had crystallized. The crystalline A-32887 was separated by filtration and dried. The A-32887 was recrystallized by dissolving in acetone (500 ml), slowly adding water (200 ml), and allowing the resulting solution to stand at room temperature. The recrystallized A-32887 was separated by filtration, washed with water and dried to give 59 g of A-32887 Na-K salt. Further recrystallization gave additional A-32887 (Na-K salt) in the following amounts: 8.4 g in the second crop, and 5.2 g in the third crop (mp 158-160°).

EXAMPLE 3 Preparation of A-32887 Free Acid

A-32887 Na-K salt (1 g), obtained as described in Example 2, was dissolved in dioxane (200 ml). Water (25 ml) was added to this solution; the resulting solution was adjusted to pH 3 by the addition of dilute HCl. The acidified solution was stirred, and maintained at pH 3 with HCl, as water (100 ml) was slowly added. The resulting solution was evaporated under vacuum to remove the dioxane; the resulting aqueous suspension was extracted twice with ethyl acetate (equal volumes). The combined ethyl acetate extract was evaporated under vacuum to give an oily residue. This residue was dissolved in chloroform and re-evaporated under vacuum to give A-32887 free acid as a white amorphous powder (524 mg; mp about 90° C.)

EXAMPLE 4 Preparation of A-32887 Silver Salt

A-32887 Na-K salt (200 mg), obtained as described in Example 2, was dissolved in methanol (10 ml). An aqueous solution of silver nitrate (2 ml; 50 mg/ml) was added slowly. The resulting solution was placed in a beaker wrapped with aluminum foil to prevent degradation (reduction) of the silver. The solution was kept at 5° C. until crystallization was complete. The crystals were separated by filtration and were recrystallized from n-hexane to give A-32887 silver salt as very fine white needles (mp 166-168° C.).

EXAMPLE 5 Preparation of the Sodium Salt of A-32887

A-32887 free acid (500 mg), prepared as described in Example 3, was dissolved in acetone (150 ml); water (20 ml) was added. The resulting mixture was adjusted to pH 9 with NaOH. Water (200 ml) was then added, and the resulting solution was stirred for 1/2 hour. The solution was concentrated under vacuum to remove acetone. The resulting suspension was extracted with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated under vacuum to dryness. The residue was dissolved in warm acetone (20 ml); water was added until the solution was turbid; and the solution was then allowed to crystallize. The crystals were removed by filtration, washed with water, and dried to give 306 mg of A-32887 sodium salt (mp 130°-133° C.).

EXAMPLE 6 Preparation of the Methyl Ether Derivative of A-32887

A-32887 free acid (3 g), prepared as described in Example 3, was dissolved in methanol (300 ml) and allowed to stand at room temperature for 12 hours. The conversion was monitored by silica-gel TLC, using a benzene:ethyl acetate (1:1) solvent system and H₂ SO₄ spray for detection. The solution was evaporated to dryness under vacuum. The residue obtained was dissolved in benzene (40 ml). This solution was applied to a 3.2- × 95-cm column of silica gel (Grace, grade 62) packed in benzene. The column was eluted with benzene:ethyl acetate (3:2), collecting 25-ml fractions, and monitoring elution by TLC. Fractions 90-220, which contained most of the desired product, were combined and evaporated under vacuum to dryness. The residue, dissolved in benzene (15 ml) was rechromatographed on a 1.8- × 112-cm column of silica gel (Grace, grade 62), prepared in benzene. The column was eluted with benzene:ethyl acetate (9:1), collecting 25-ml fractions and monitoring elution by TLC. At fraction 383, the eluting solvent was changed to benzene:ethyl acetate (4:1); and at fraction 780 the solvent was changed to benzene:ethyl acetate (7:3). Fractions 450-700 contained A-32887; fractions 702-760 contained a mixture of A-32887 and A-32887 methyl ether derivative; and fractions 761-1130 contained A-32887 methyl ether derivative. Fractions 761-1130 were combined and evaporated to dryness under vacuum. The residue was dissolved in ethyl acetate (20 ml); n-hexane (80 ml) was added; and the solution was allowed to crystallize. The crystals were removed by filtration and dried to give 242 mg of A-32887 methyl ether derivative as the sodium salt (mp 214°-216° C.).

EXAMPLE 7 Preparation of the Acetyl Ester Derivative of A-32887

A-32887 Na-K salt (200 mg), prepared as described in Example 2, was dissolved in pyridine (8 ml); acetic anhydride (3.2 ml) was added. The mixture was allowed to stand overnight and then was evaporated under vacuum to dryness. The residue was dissolved in t-butanol and lyophilized to give 231 mg of the acetyl ester derivative of A-32887 (Na-K salt) as a white amorphous powder, mp 127°-129° C.

EXAMPLE 8 Preparation of the n-Butyryl Ester Derivative of A-32887

A-32887 (Na-K salt; 200 mg) was dissolved in pyridine (14 ml), and n-butyric anhydride (14 ml) was added. The mixture was allowed to stand for 17 hours at room temperature; water (14 ml) was added; and the solution was then concentrated to an oil in vacuo. The oily residue was lyophilized from dioxane-water several times to give 240 mg of the n-butyryl ester derivative of A-32887 (Na-K salt) as a white amorphous powder, mp 59°-62° C.

EXAMPLE 9 A-32887-Modified Chick Ration for Coccidiosis Control

A balanced, high-energy ration adapted to feed chicks for rapid weight gain is prepared by the following recipe:

    ______________________________________                                         Ingredient          %        lbs                                               ______________________________________                                         Ground yellow corn  50       1,000                                             Soybean meal, solvent-                                                         extracted dehulled, finely                                                     ground, 50 percent protein                                                                         31.09    621.8                                             Animal fat (beef tallow)                                                                           6.5      130                                               Dried fish meal, with                                                          solubles (60% protein)                                                                             5.0      100                                               Distillers' solubles                                                           from corn           4.0      80                                                Dicalcium phosphate,                                                           feed grade          1.8      36                                                Calcium carbonate   0.8      16                                                Vitamin premix                                                                  (representing vitamins A,                                                      D, E, K, and B.sub.12, choline,                                                niacin, pantothenic acid,                                                      riboflavin, biotin, with                                                       glucose bulking agent)                                                                            0.5      10                                                Trace mineral premix                                                            (representing MnSO.sub.4, ZnO,                                                 KI, FeSO.sub.4, CaCO.sub.3)                                                                       0.2      4                                                 2-Amino-4-hydroxybutyric                                                       acid                                                                            (hydroxy analog of                                                             methionine)        0.1      2                                                 A-32887 (Na-K Salt) 0.01     0.2                                               ______________________________________                                    

These substances are mixed in accordance with standard feed-mixing techniques. Chicks fed such a ration, with water ad libitum, are protected against exposure to coccidiosis; weight gains are comparable to those of coccidiosis-free chicks fed a similar, unmedicated diet.

EXAMPLE 10 A-32887-Improved Beef-Cattle Ration

A balanced high-grain beef-cattle ration is prepared as follows:

    ______________________________________                                         Ingredient         %          lbs                                              ______________________________________                                         Finely ground corn 67.8       1356                                             Ground corn cob    10         200                                              Dehydrated alfalfa meal,                                                       17 percent protein 5          100                                              Dehulled soybean meal,                                                         solvent extracted,                                                             50 percent protein 9.9956     199.912                                          Cane molasses      5          100.0                                            Urea               0.6        12.0                                             A-32887 (Na-K salt)                                                                               0.0044     0.088                                            Dicalcium phosphate,                                                           feed grade         0.5        10.0                                             Calcium carbonate  0.5        10.0                                             Sodium chloride    0.3        6.0                                              Trace mineral premix                                                                              0.03       0.6                                              Vitamin A and D.sub.2 premix*                                                                     0.07       1.4                                              Vitamin E premix** 0.05       1.0                                              Calcium propionate 0.15       3.0                                              ______________________________________                                          *Containing per pound: 2,000,000 I.U. of vitamin A; 227,200 I.U. of            vitamin D.sub.2 and 385.7 g. of soybean feed with 1% oil added                 **Corn distillers dried grains with solubles containing 20,000 I.U. of         d-alpha-tocopheryl acetate per pound                                     

The mixed feed is compressed into pellets. At an average daily ingestion rate of 15 pounds of feed per animal, this feed supplies approximately 300 mg. of A-32887 (Na-K salt) per animal per day.

EXAMPLE 11 A-32887-Improved Swine Ration

A balanced swine ration is prepared as follows:

    ______________________________________                                         Ingredient            %        lbs./ton                                        ______________________________________                                         Corn, Yellow, Ground  73.15    1463                                            Soybean Oil Meal, Solvent                                                      Extracted, Dehulled, 50%                                                                             12.30    246                                             Alfalfa Meal, Dehydrated, 17%                                                                        2.50     50                                              Meat Scraps, 55%      2.50     50                                              Fish Meal             2.50     50                                              Distiller Dried Solubles (Corn)                                                                      2.50     50                                              Animal Fat            2.00     40                                              Calcium Carbonate     0.70     14                                              Dicalcium Phosphate, Feed Grade                                                                      0.50     10                                              NaCl                  0.50     10                                              Swine Vitamin Premix.sup.1                                                                           0.50     10                                              Methionine Hydroxy Analog, 93%                                                                       0.20     4                                               Trace Mineral Premix.sup.2                                                                           0.10     2                                               Selenium Premix.sup.3 0.05     1                                               Total                 100.00   2000                                            ______________________________________                                          .sup.1 Each kg of premix contains the following: 77,161 IU Vitamin D.sub.      ; 2,205 IU Vitamin E; 411 mg riboflavin; 1,620 mg pantothenic acid; 2,205      mg niacin; 4.4 mg Vitamin B.sub.12 ; 441 mg Vitamin K; 19,180 mg choline;      110 mg folic acid; 165 mg pyridoxine; 110 mg thiamine; 22 mg biotin.           .sup.2 Each kg of premix contains the following: 50 g manganese as             manganese sulfate; 100 g zinc as zinc carbonate; 50 g iron as ferrous          sulfate; 5 g copper as copper oxide; 1.5 g iodine as potassium iodide and      150 g maximum and 130 g minimum calcium as calcium carbonate.                  .sup.3 Each kg of premix contains 200 mg of selenium as sodium selenite. 

A-32887 (Na-K salt; 100 g) is mixed with from about 5 to about 10 lbs. of soybean mill run to give a feed premix. From 5-10 lbs of this A-32887 premix is thoroughly mixed with a sufficient amount of the above-described swine ratio to give a concentration of 100 g of A-32887 per ton of ration. Swine fed such a ration, with water ad libitum, are protected against the lethal effects of swine dysentery. 

We claim:
 1. A method of increasing feed-utilization efficiency in ruminant animals which comprises orally administering to an animal in need thereof an effective propionate-increasing amount of a compound selected from the group consisting of (1) antibiotic A-32887, which in its free acid form is a white amorphous powder melting at about 90° C. and having the following characteristics:(a) a molecular weight of about 946, as determined by mass spectrometry; (b) an approximate empirical formula of C₄₈₋₄₉ H₈₀₋₈₆ O₁₇₋₁₈ and a preferred empirical formula of C₄₈ H₈₂ O₁₈ ; (c) an approximate percentage elemental composition of 61.61% carbon, 8.56% hydrogen, and 28.63% oxygen; (d) an infrared absorption spectrum in chloroform with significant absorption maxima at the following frequencies (cm⁻¹): 3540 (shoulder), 3420 (medium), 2945 (shoulder), 2905 (strong), 2860 (shoulder), 2805 (shoulder), 1710 (weak), 1450 (medium), 1370 (medium), 1350 (shoulder), 1300 (shoulder), 1275 (weak), 1170 (shoulder), 1155 (medium), 1100 (shoulder), 1085 (strong), 1060 (strong), 1010 (weak), 990 (weak), 980 (shoulder), 948 (medium), 890 (weak), and 850 (weak); (e) no significant absorption in the ultraviolet spectrum; (f) a proton-magnetic-resonance spectrum which indicates the presence of five methoxyl groups; (g) a specific rotation as follows: [α]_(D) ²⁵ + 15.9° (c 1, CHCl₃); (h) an acid function capable of forming salts and ester derivatives; (i) at least one hydroxyl group capable of esterification;and which in its mixed sodium-potassium salt form is a white crystalline compound, when crystallized from acetone-water, having the following characteristics: (a') a melting point of about 187°-190° C.; (b') an approximate percentage elemental composition of 60.14% carbon, 8.11% hydrogen, 29.64% oxygen, 2.31% sodium, and 0.46% potassium; (c') an infrared absorption spectrum in chloroform with significant absorption maxima at the following frequencies (cm⁻¹): 3400 (shoulder), 3210 (medium), 2970 (strong), 2925 (strong), 2870 (weak), 2820 (weak), 1605 (medium), 1455 (medium), 1375 (shoulder), 1358 (medium), 1310 (weak), 1285 (weak), 1183 (medium), 1160 (medium), 1110 (shoulder), 1090 (strong), 1060 (strong), 1012 (weak), 980 (medium), 942 (medium), 910 (weak), 875 (shoulder), and 858 (weak); (d') an X-ray powder diffraction pattern (CuNi, 1.5405 λ, d=interplanar spacing in angstroms) as follows:

    ______________________________________                                                             Relative                                                   d                   Intensity                                                  ______________________________________                                         12.53               50                                                         10.10               100                                                        9.75                40                                                         9.05                40                                                         8.07                70                                                         7.16                100                                                        6.77                100                                                        6.48                70                                                         6.16                60                                                         5.58                50                                                         5.35                50                                                         5.10                30                                                         4.86                60                                                         4.70                50                                                         4.44                50                                                         4.25                50                                                         4.06                40                                                         3.80                30                                                         3.68                40                                                         3.58                30                                                         3.42                60                                                         3.22                10                                                         3.06                10                                                         2.97                10                                                         2.85                10                                                         2.76                02                                                         2.61                15                                                         2.49                05                                                         2.47                05                                                         2.44                15                                                         2.36                10                                                         2.29                10                                                         2.20                02                                                         2.12                02                                                         2.03                05                                                         1.96                05                                                         1.90                02                                                         1.81                02                                                         ______________________________________                                    

(e') a specific rotation as follows: [α]_(D) ²⁵ + 9.6° (c 1, CHCl₃); (f') a titratable group in 80% aqueous dimethylformamide with a pKa value of 4.60; (g') solubility in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; slight solubility in hexane; and insolubility in water; (h') stability in aqueous solutions having a pH of from about 3 to about 11, instability in solutions having a pH lower than about 3; (i') approximate R_(f) values in the following paper-chromatographic systems using Bacillus subtilis ATCC 6633 bioautography for detection:

    ______________________________________                                         Solvent System             R.sub.f Value                                       ______________________________________                                         Water saturated with methyl                                                    isobutyl ketone (MIBK)     0.58                                                Water:methanol:acetone                                                         (12:3:1). This solution                                                        is adjusted to pH 10.5     with                                                NH.sub.4 OH and then lowered                                                   to pH 7.5 with H.sub.3 PO.sub.4                                                                           0.32                                                Propanol:water (1:9)       0.85                                                Methanol:propanol:water (6:2:1).                                               Paper buffered with 0.75 M KH.sub.2 PO.sub.4,-pH 4.0.                                                     0.79                                                Methanol:0.05 M sodium citrate at                                              pH5.7 (7:3). Paper buffered with                                               0.05 M sodium citrate at pH 5.7.                                                                          0.85                                                Propanol:water (7:3)       0.91                                                ______________________________________                                    

(j') approximate R_(f) values in the following thin-layer chromatographic systems, using either vanillin spray reagent or Bacillus subtilis bioautography for detection:

    ______________________________________                                         Solvent System             R.sub.f Value                                       ______________________________________                                         Methanol                   0.80                                                Ethyl acetate:ethanol (1:4)                                                                               0.65                                                Ethyl acetate:chloroform (1:1)                                                                            0.36                                                Ethyl acetate:chloroform (6:1)                                                                            0.67                                                Benzene:ethyl acetate:methanol                                                 (6:4:0.2)                  0.58                                                ______________________________________                                    

and which in its sodium salt form has a molecular weight of about 968 and in its potassium salt form has a molecular weight of about 984, both as determined by FD mass spectrometry; and which in its silver salt form is a crystalline compound (from n-hexane) with a melting point of 166°-168° C.; (2) the acetyl ester derivative of antibiotic A-32887, having an approximate empirical formula of C₅₀₋₅₁ H₈₂₋₈₈ O₁₈₋₁₉, and which in its Na-K salt form has a molecular weight of about 988, a melting point of about 127°-129° C., and an approximate R_(f) value of 0.40 on silica-gel TLC in benzene:ethyl acetate (1:1), and an infrared absorption spectrum as shown in FIG. 4 of the drawings; (3) the n-butyryl ester derivative of antibiotic A-32887, having an approximate empirical formula of C₅₂₋₅₃ H₈₆₋₉₂ O₁₈₋₁₉, and which in its Na-K salt form has a molecular weight of about 1016, a melting point of about 59°- 62° C., an approximate R_(f) value of 0.64 on silica-gel TLC in benzene:ethyl acetate (1:1), and an infrared absorption spectrum as shown in FIG. 5 of the drawings; and (4) the pharmaceutically-acceptable salts thereof.
 2. The method of claim 1 wherein the compound is antibiotic A-32887 or a pharmaceutically-acceptable salt thereof.
 3. The method of claim 2 wherein the compound is antibiotic A-32887 Na-K salt.
 4. A method of increasing feed-utilization efficiency in ruminant animals which comprises orally administering to an animal in need thereof an effective propionate-increasing amount of a compound selected from the group consisting of (1) the methyl ether derivative of A-32887, having a molecular weight of about 960 and an approximate empirical formula of C₄₉₋₅₀ H₈₂₋₈₈ O₁₇₋₁₈, and which in its sodium salt form is a white crystalline compound, when crystallized from n-hexane: ethyl acetate, having a melting point of about 214°-216° C. and having the following characteristics:(a) a molecular weight of about 982, as determined by FD mass spectrometry; (b) an infrared absorption spectrum in chloroform with significant absorption maxima at the following frequencies (cm⁻¹): 3400 (broad), 2990, 2960, 2930, 2870, 2820, 1725, 1610, 1455, 1407, 1370, 1309, 1282, 1240, 1180, 1158, 1110, 1093, 1082, 1057, 1010, 980, 945, 900, 868, 858, 827, 802, 700, and 653; (c) a proton-magnetic-resonance spectrum which indicates the presence of six methoxyl groups; (d) a specific rotation as follows: [α]_(D) ²⁵ - 5.1° (c 1, CHCl₃); (e) an X-ray powder diffraction pattern (CuNi, 1.5405 λ, d = interplanar spacing in angstroms) as follows:

    ______________________________________                                                             Relative                                                          d            Intensity                                                  ______________________________________                                                13.18        50                                                                12.01        50                                                                9.02         100                                                               8.26         100                                                               7.19         80                                                                6.46         50                                                                5.78         20                                                                5.46         20                                                                5.00         30                                                                4.24         20                                                                3.72         20                                                                3.36         05                                                         ______________________________________                                    

(f) a titratable group in 80% aqueous dimethylformamide with a pK_(a) value of about 5.4; (g) solubility in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; slight solubility in hexane and heptane; and insolubility in water; (h) an acid function capable of forming salts and ester derivatives; and(2) the pharmaceutically-acceptable salts of the A-32887 methyl ether derivative.
 5. The method of claim 4 wherein the compound is A-32887 methyl ether derivative sodium salt. 